Abstract

Defence against Leishmania depends upon Th1 inflammatory response and, a major problem in susceptible models, is the turnoff of the leishmanicidal activity of macrophages with IL-10, IL-4, and COX-2 upregulation, as well as immunosuppressive PGE2, all together inhibiting the respiratory burst. Peroxisome proliferator-activated receptors (PPAR) activation is responsible for macrophages polarization on Leishmania susceptible models where microbicide functions are deactivated. In this paper, we demonstrated that, at least for L. mexicana, PPAR activation, mainly PPAR γ , induced macrophage activation through their polarization towards M1 profile with the increase of microbicide activity against intracellular pathogen L. mexicana. PPAR activation induced IL-10 downregulation, whereas the production of proinflammatory cytokines such as TNF- α , IL-1 β , and IL-6 remained high. Moreover, PPAR agonists treatment induced the deactivation of cPLA2-COX-2-prostaglandins pathway together with an increase in TLR4 expression, all of whose criteria meet the M1 macrophage profile. Finally, parasite burden, in treated macrophages, was lower than that in infected nontreated macrophages, most probably associated with the increase of respiratory burst in these treated cells. Based on the above data, we conclude that PPAR agonists used in this work induces M1 macrophages polarization via inhibition of cPLA2 and the increase of aggressive microbicidal activity via reactive oxygen species (ROS) production.

PPAR agonists inhibit cPLA2 phosphorylation and COX-2 expression in L. mexicana-infected macrophages. (a) Protein expression for PPARs and COX-2 and cPLA2 phosphorylation levels were evaluated by Western blotting. (b) Densitometry analyses of cPLA2 phosphorylation and (c) COX-2 expression were performed in basal conditions as well as in macrophages treated or not with PPAR agonists. (d) COX-2 mRNA expression was evaluated by qRT-PCR and analyzed by 2−ΔΔCT method. Total ERK1/2 was probed to normalize protein loading. Results are representative of three independent experiments. Graph bars are mean ± SEM of three independent experiments, and statistical analysis was done comparing, for each time, treated versus nontreated macrophages; (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.

Cytokine determination in L. mexicana-infected macrophages. Levels of gene expression for each sample were normalized with β-actin RNA as internal control. Modulation was expressed relative to the untreated control using the 2−ΔΔCT method. The x-axis intercepts the y-axis at “1” to show the increase and the decrease of each cytokine compared to nontreated infected macrophages. Relative expression level for each cytokine was calculated according to ΔΔCT = (CT test − CTβ-actin) treated − (CT test − CTβ-actin) untreated formula []. Graph bars are mean ± SEM of three independent experiments, and statistical analysis was done comparing, for each time, treated versus nontreated macrophages; (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.

TLR4 expression in L. mexicana-infected macrophages. Cells were treated or not with PPAR agonists for 24 h and cPLA2 antagonist for 1 h before infection. TLR4 expression was analyzed by flow cytometry. LPS (2 h) was used as a positive control of induction. Graph bars are mean ± SEM of three independent experiments, and statistical analysis was performed comparing, for each time, treated versus non-treated macrophages; (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.

Prostaglandin production by L. mexicana-infected macrophages. Prostaglandins were analyzed by MS/MS assay; product scanning experiments were conducted using nitrogen as collision gas, and the collision energy was optimized for individual compounds to generate the most abundant product ions. These product ion spectra were then used to select the precursor-product ion pairs for the development of MRM assays. Deuterium-labeled prostaglandins were used as internal standards for quantitation. Graph bars are mean ± SEM of three independent experiments, and statistical analysis was done comparing, for each time, treated versus nontreated macrophages; (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.

Oxidative burst of L. mexicana-infected macrophages. Cells were treated with PPAR agonists 24 h before infection, and the oxidative burst was determined by NBT reduction. NBT was added simultaneously with promastigotes. (a) After the indicated times after infection, slides with infected macrophages were washed and stained for 30 min with Fuccina. Microphotographs show positive cells to NBT reduction in comparison with control cells, which were treated or not with agonists in the presence of NBT. (b) The graph shows percentage of cells positive to NBT reduction. (c) Quantitative analysis of NBT reduction of macrophages infected and treated or not with PPAR agonists. Graph bars are mean ± SEM of three independent experiments, and statistical analysis was done comparing, for each time, treated versus nontreated macrophages; (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.